FIGURE 1.

Visualization of endogenous mRNAs encoding mitochondrial proteins. (A) mMP colocalization with mitochondria. Cells bearing the MS2L sequence integrated into different ORFs were transformed with plasmids expressing MS2-CP-GFP(x3) and Oxa1-RFP (a mitochondrial marker). Cells were grown on liquid synthetic medium containing glucose to mid-log phase at 26°C and were induced with the same medium lacking methionine for 1 h prior to visualization by confocal microscopy. For the CIT1, DNM1, and MDM10 mRNAs, the cells were grown on glycerol (2%)–containing medium instead of glucose as the carbon source. The percentage of fluorescent RNA granules that colocalized with mitochondria (% Loc.) is given in %. (mRNA) Indicates the localization of GFP-labeled RNA granules. (Mitochondria) Indicates the localization of mitochondria labeled with Oxa1-RFP. (Merge mRNA/Mit) Indicates merger of the mitochondrial and mRNA windows. [Merge (all)] Indicates merger of the light (DIC), mitochondrial, and mRNA windows. Scale bar, 2 μm. (B) Chromosomal MS2 aptamer gene-tagging does not affect protein function. Wild-type, puf3Δ, ATP2::loxP::MS2L::ATP2^3′UTR (ATP2_INT), atp2Δ, OXA1::loxP::MS2L::OXA1^3′UTR, and oxa1Δ cells were grown to mid-log phase at 26°C on glucose-containing medium, serially diluted, plated onto solid synthetic medium containing either glucose (Glu) or glycerol (Gly) as a carbon source, and grown for 48 h at 26°C. Note that functional Oxa1 and Atp2 are necessary for growth on glycerol.